PROJECT SUMMARY/ABSTRACT Aging represents a progressive loss of tissue function over time resulting in an exponentially increasing mor- tality rate. While aging itself is not recognized as a disease, it is considered the single greatest predisposing factor for a myriad of age-related diseases, including atherosclerosis, diabetes, arthritis, and cancer. Therefore, therapies that attenuate fundamental drivers of aging hold great promise for preventing numerous age-related diseases and reducing all-cause mortality. One potential mechanism of aging is cellular senescence, a com- plex tumor-suppressive cell fate involving a permanent cell-cycle arrest and a distinctive secretome, which in- cludes inflammatory cytokines, growth factors and matrix metalloproteinases. While a link between aging and senescent cells has been hypothesized since their discovery, the perception that these cells are viable thera- peutic targets is a recent phenomenon originating from our studies showing that clearance of senescent cells improves healthspan and lifespan in mice. Our long-term objective is to discover senescent cell-focused strat- egies for healthy aging based on a solid foundation of basic knowledge. The objective of this application is therefore to answer fundamental questions regarding senescent cells that accumulate in vivo in mammals. These include which cell types become senescent in different tissues and how, and what the molecular proper- ties and biological effects of the various senescent cell populations are. The critical first step towards realizing these goals is to overcome longstanding technical barriers for the detection and collection of naturally occur- ring and disease-associated senescent cells. In preparation for this proposal we have worked to remove these barriers by establishing powerful new methods and a series of genetically engineered mouse models. Based on published and preliminary data, our central hypothesis is that senescent cells arising with aging and age- related disease are heterogeneous, with their diverse effects in vivo resulting from differences in senescence- inducing stressors, cell type origins and gene transcription profiles. We will test this hypothesis and accomplish the objectives of this application by pursuing two interrelated aims that are comprehensive, largely complemen- tary, and not interdependent. In Aim 1, we will characterize the properties of senescent cells that accumulate with aging and disease using a newly generated knockin mouse strain that allows for the detection, tracking and collection of naturally occurring senescent cells residing in aged or diseased tissues. In Aim 2, we will dis- sect the local versus systemic detrimental effects of senescent cells in aging or disease phenotypes through two complementary approaches involving newly generated mouse strains that allow for tissue-specific elimina- tion or generation of senescent cells. In addition to providing important new fundamental insights into how se- nescent cells contribute to the biology of aging, we expect these experiments to have an impact from a thera- peutic perspective as data obtained will provide information about the effectiveness of local versus systemic senescent cell elimination in delaying aging-related phenotypes globally and in a particular tissue.